1. Field of the Invention
This invention lies in the field of in vitro techniques and compositions for replenishing fluid electrolytes and nutrients while regulating metabolic processes in living animal cells.
2. State of the Art
The vital functions of highly developed organisms are closely dependent on the internal aqueous medium and on the maintenance in it of extreme constancy of chemical and physical properties.
Typically in vitro fluids of this type are aqueous electrolyte solutions which are used to contact living cells in, for examples, (1) incubation of tissue slices, minces or homogenates, (2) perfusion of isolated organs as kidney's, liver, muscles, or heart, (3) incubations of isolated cell suspensions such as isolated adipocytes, hepatocytes, blood cells, myocytes etc., and (4) most particularly, electrolytes or "balanced salt mixtures" in which cells in culture are grown after the optional addition thereto of a host of various nutrients, such as vitamins, sugars, amino acids, hormones, and the like. As will be shown all of other famous solutions, e.g., Hank's (Proc Soc Exp Biol Med 71: 196, 1949), Delbecco's (J Exp Med 99: 167-182, 1954), Earle's (J Nat'l Canc Inst 4: 165-212, 1943), etc., used in the tissue culture art are very simple variations of only 2 basic electrolyte solutions--usually Krebs-Henseleit (Krebs H A, Henseleit K A. Hoppe-Seyler's Z Physiol 210:33-66, 1932) with variations for the excessive Ca.sup.2+ used by Krebs, and Krebs-ringer-Phosphate (Krebs H A. Hoppe-Seyler's Z Physiol Chem 217: 193, 1933), where for convenience of the Experimenter buffering of the pH is achieved with excessive Pi (inorganic phosphate) rather than with HCO.sub.3.sup.- /CO.sub.2.
In this disclosure we propose the first major basic advance in these fundamental (basic) electrolyte solutions for the in vitro art since Kreb's attempted to correct the abnormal Na:Cl ratio present in all such solutions in 1950 (Krebs H A. Biochem Biophys Acta 4: 249-269, 1950).
It has long been recognized that all animal intracellular and extracellular body fluids contain inorganic electrolytes, and that these electrolytes are involved in, and profoundly influence, various life processes. Attempts to make artifical electrolyte fluids which may bathe tissues or be administered to the human blood stream have been known since about 1880, and, although modern analytical tools and procedures have clarified compositional details of blood electrolytes, the use of varous aqueous electrolyte solutions for in vitro purposes in tissue culture, organ perfusion and related fields has long been extant.
Those inorganic electrolytes characteristically found in normal human blood serum at respective concentration levels above about 1 millimolar per liter of concentration are shown below in Table I. Also, for comparative purposes, in Table I are shown some representative compositions of various aqueous electrolyte solutions that have been previously prepared and used for in vitro purposes. In general, the philosophy behind the formaluation of aqueous electrolyte solutions for in vitro use has been that such should mimic or closely resembled the chemical composition of electrolytes in blood, (plasma) extracellular fluids and intracellular fluids. An electrolyte is a substance (usually a salt, acid or base) which in solution dissociates wholly or partly into electrically charged particles known as ions (the term is also sometimes used inthe art to denote the solution itself, whichhas a higher electrical conductivity than the pure solvent, e.g. water). The positively charged ions are termed cations while the negatively charged ions are termed anions. Strong and weak electrolytes are recognized. The dissociation of electrolytes is very markedly dependent on concentration; it increases with increasing dilution of the solution. The ions can be regarded as molecules in electrolyte solution. Because of dissociation considerations, the term "sigma" or the greek letter for sigma (".SIGMA.") is sometimes employed herein as a prefix to designate the total presence of a specified material, such as an electrolyte, whether or not all of the material is in an ionic form complexed with a heavy metal, or regardless of charge on the material in a given solution. A pair of brackets [ ] indicates the free concentration of the substance indicated as opposed to that bound to tissue components, such as proteins. TBL3 TABLE I Prior Art Simulated Plasma Electrolyte Solutions for Contacting Living Cells in Vitro. (14) Normal (1) (2) (4) (13) Krebs Plasma Normal Normal (3) Mammalian (5) (6) (11) (12) Krebs Improved Units N.E.J.M. Saline Saline Ringer's Ringer's Lactated Lactated (7) (10) Krebs Krebs Improved Ringer III mmoles 283, 1285 0.9% NaCl 0.95% NaCl Injection U.K. Ringer's Ringer's Acetated (8) (9) Krebs Ringer Serum Ringer II Low HCO.sub.3.sup.- L fluid 1970 U.S. U.K. U.S. & Canada U.S. (Hartmann) Ringer's Locke's Tyrode's Henseleit Phosphate Substitute Ca.sup.2+ free Low Pi Na 136-145 154 162.5 147 156.4 130 129.8 130 157.57 150.1 143 150.76 140.31 147.41 140.86 K 3.5-5.0 4 5.4 4 5.4 4 3.57 5.9 5.9 5.92 5.92 5.92 5.92 Ca 2.1-2.6 2.5 1.15 1.5 0.9 1.5 2.16 1.8 2.5 2.54 2.54 2.54 free [Ca.sup.2+ ] [1.06] Mg 0.75-1.25 1.0 0.45 1.2 1.18 1.18 1.18 1.18 free [Mg.sup.2+ ] [0.53] .SIGMA. mEq Cations 142.7-153.2 154 162.5 156 164.1 137 139 137 165.46 160.5 156.3 164.12 153.7 155.69 154.22 Cl 100-106 154 162.5 156 161.7 109 111.8 109 163.92 147.48 127.8 131.51 104.62 103.06 122.36 HCO.sub.3 26-28 2.4 3.57 11.9 25 24.9 3.56 3.56 .SIGMA. Pi 1-1.45 1.22 1.18 17.38 1.18 15.03 3.49 SO.sub.4 0.32-0.94 1.18 1.18 1.18 1.18 1.18 L-lactate 0.6-1.8 28(d,l) 27.2(d,l) pyruvate 4.92 4.92 4.92 Lact/pyr D-.beta.-OHbutyrate acetoacetate .beta. HB/acac acetate 28 Other fumarate.sup.2- 5.39 5.39 5.39 glutamate.sup.- 4.92 4.92 4.92 .SIGMA. mEq anions 128.7-139.4 154 162.5 156 164.1 137 139 137 167.49 161.6 157.3 163.97 152.49 156.64 155.17 Na/Cl 1.28-1.45 1.00 1.00 0.94 0.97 1.23 1.16 1.19 0.96 1.02 1.12 1.15 1.34 1.43 1.15 Glucose 3.9-5.6 5.6-13.9 5.6 11.5 11.5 11.5 or others CO.sub.2 0.99-1.39 1.24 1.24 pH 7.35-7.45 .apprxeq.5.5-6.5 .apprxeq.5.5-6.5 .apprxeq.5.5-6.5 .apprxeq.7.0 7.1 7.4 7.4 7.4 .apprxeq.7.6 .apprxeq.7.6 .SIGMA. mOsm 285-295 308 325 309 324 272.5 276 272.5 336 318.8 308 311.7 309.8 304.1 307.8 (1) Usual "physiological saline" in the U.S. is a 0.9% or 154 mM. (Gilman AG, Goodman LS, Gilman A. The Pharmacological Basis of Theraputics (1980) pp 848-884, McMillan, London. (2) "Physiological Saline" in the U.K. is 0.95% NaCl. (Diem K. ed. Documenta Geigy (1962) pp 333-334, Geigy, Manchester. (3) All "Ringer's solutions" are derived from Ringer S. Physiol 4, 29, & 222, 1893 and 7, 1886. This commercial U.S. version is from Facts and Comparisons, Oct 1981, p. 50, Lippincott, St. Louis. (4) From Best and Taylor, Physiological Basis of Medical Practice, 6th edition, Baltimore, 1950. (5) From Facts and Comparisons p. 50, Oct '81, Lippincott, St. Louis. (6) Hartmann AF. J Am Med Assoc 103: 1349-1354, 1934. (7) Fox CL et al. J Am Med Assoc 148: 825-833, 1952. (8) Locke FS. Zbl Physiol 8,166, 1894; 14, 670, 1900; 15, 490, 1901. (9) Tyrode MJ. Arch int Pharmacodyn 20, 205, 1910. (10) Krebs HA, Henseleit KA. HoppeSeyle's Z Physiol Chem 210, 33-66, 1932 (11) Krebs HA. HoppeSeyle's Z Physiol Chem 217, 193, 1933. (12)-(14) Krebs HA. Biochem Biophys Acta 4, 249-269, 1950.
TABLE II __________________________________________________________________________ " Prior Art Perfusion Fluids" (15) Krebs Normal Liver Per- Plasma fusion with (16) (17) (19) (20) Units N.E.J.M. Bovine serum Schimassek Krebs (18) Bahlman Fulgraff mmoles 283, 1285 Albumin and Liver Kidney Hepatocyte Kidney Kidney L fluid 1970 Red Cells Perfusion Perfusion Incubation Perfusion Perfusion __________________________________________________________________________ Na 136-145 153 151.54 148 153 147 143 K 3.5-5.0 5.9 5.9 5.9 5.9 4.9 4.74 Ca 2.1-2.6 2.5 1.8 2.5 2.5 2.56 1.25 free [Ca.sup.2+ ] [1.06] Mg 0.75-1.25 1.2 0.49 1.2 1.2 1.2 0.59 free [Mg.sup.2+ ] [0.53] .SIGMA. mEq Cations 142.7-153.2 166.3 162.02 161.3 166.3 159.4 151.15 Cl 100-106 127.8 147.48 127.8 127.8 127 113.04 HCO.sub.3 26-28 25 11.9 25 25 24.5 25 .SIGMA. Pi 1-1.45 1.18 1.22 1.18 1.18 1.18 1.18 SO.sub.4 0.32-0.94 1.18 -- 1.2 1.2 1.18 1.18 L-lactate 0.6-1.8 (10Na-1Lac) 1.33 5Na--1Lac 9.09 2.75(d,l) 3.5(?d,l) pyruvate 0.09 0.91 0.25 0.25 Lact/pyr 14.8 10 10 7 or 14 D-.beta.-OHbutyrate acetoacetate .beta.-HB/acac acetate 5.0 Other .SIGMA. mEq anions 128.7-139.4 167.0 162.81 162.3 167.0 159.1 151.31 Na/Cl 1.28-1.45 1.12 1.03 1.16 1.20 1.20 1.26 (1.20) Glucose 3.9-5.6 5.45 6.2 -- or others 6.7 urea 6.7 urea CO.sub.2 0.99-1.39 1.25 1.24 1.24 1.24 1.24 1.24 pH 7.35-7.45 7.4 7.1 7.4 7.4 7.4 7.4 .SIGMA. mOsm 285-295 328 321 318 328 327 307.9 Albumin (g %) 3.5-5 3.9 2.5 5 2.5 5.5 0.05 __________________________________________________________________________ *Artificial perfusion fluid generally add 1.5 to 8 g % albumin, dialyzed against a medium listed in Table I; that is KrebsHenseleit (10), KrebsRinger Phosphate (11), Tyrode's (9), Locke's (8), or KrebsHenseleit with a lowered Ca.sup.2+ to the 1 mM range, particularly in heart perfusion. They may or may not contain red cells. KrebsHenseleit is known to contain about twice the amount of ionized Ca.sup.2+ as serum. (15) Hems R, Ross BD, Berry MN, Krebs HA. Biochem J 101, 284, 1966; Krebs Henseleit (10) with 3.9 g % bovine albumin. (16) Schmassek H. Biochem Z 336, 460, 1963. Essentially Tyrode's (9) with added lactate and pyruvate. (17) NishiitsutsujiUwo JM, Ross BD, Krebs HA. Biochem J 103, 852-862, 1967; KrebsHenseleit (10) with 5 g % albumin, dry. (18) Crow KE, Cornell NW, Veech RL. Biochem J 172, 29-36, 1978, KrebsHenseleit (10) with 2.5 g % dialysed albumin plus 1lactate plus pyruvate. (19) Bahlman J. et al. Am J Physiol 212, 77 1967; Krebs Henseleit (10) with lactate and pyruvate and 5.5 g % bovine albumin. (20) Fulgraff et al. Arch int Pharmacodyn 172, 49, 1972; KrebsHenseleit (10) with 1/2 Mg and Ca plus lactate and pyruvate, plus 5 mM acetate, plu 0.05 g % albumin plus 2 g % hemocel.
TABLE III __________________________________________________________________________ "Balanced Salt Mixtures" for Tissue Culture to Which are Added Complex Combined Nutrients of Carbohydrates, Vitamins, Amino Acids and Organic Acids as in Eagle's Basal Media (Eagle HJ. J Biol Chem 214, 839, 1955) which is Added to Earle's Salt Mixture. Normal Plasma (22) (26) (26a) (11) Units N.E.J.M (10) Earle's (24) (25) Delbecco's Delbecco's Krebs mmoles 283, 1285 Krebs Balanced (23) Hank's Ham's Phosphate Modified Ringer L fluid 1970 Henseleit Salts William's Salts F-12 Saline Eagle's Phosphate __________________________________________________________________________ Na 136-145 143 142 144.3 142.8 146.9 152.2 154.5 150.76 K 3.5-5.0 5.9 5.4 5.4 5.8 3.0 4.17 5.4 5.92 Ca 2.1-2.6 2.5 1.8 1.81 1.3 0.3 0.9 1.8 2.54 free [Ca.sup.2+ ] [1.06] Mg 0.75-1.25 1.2 0.8 0.81 0.8 0.6 0.49 0.8 1.18 free [Mg.sup.2+ ] [0.53] .SIGMA. mEq Cations 142.7-153.2 156.3 152.6 154.9 152.8 151.7 159.15 165.1 164.12 Cl 100-106 127.8 126.2 126 146 133.6 140.5 118.5 131.51 HCO.sub.3 26-28 25 23.8 26 4.17 14 -- 44 -- .SIGMA. Pi 1-1.45 1.18 1 1 0.8 1 9.83 1 17.38 SO.sub.4 0.32-0.94 1.18 0.8 0.8 0.8 0.6 0.48 0.8 1.18 L-lactate 0.6-1.8 pyruvate 0.23 0.9 1.0 Lact/pyr 0 0 0 Other .SIGMA. mEq anions 128.7-139.4 157.3 153.4 155.7 152.9 151.5 159.18 166.1 163.97 Na/Cl 1.28-1.45 1.12 1.12 1.15 0.975 1.10 1.08 1.30 1.15 Glucose 3.9-5.6 -- 5.6 11.1 5.6 10 -- 25 -- or others CO.sub.2 0.99-1.39 1.24 1.24 1.24 -- 1.24 -- 1.24 -- pH 7.35-7.45 7.4 7.4 7.4 .apprxeq.7.6 7.1 7.4 7.65 7.4 .SIGMA. mOsm 285-295 308 311 321 308.2 312 308 354 311.7 Use: Tissue Same as Tissue Tissue Tissue Tissue Culture 22 culture Culture Culture Culture Salts to manipu- which lation. nutrients are added __________________________________________________________________________ (10) It can be seen that Earle's Balanced Salts and "Williams" are just KrebsHenseleit with Mg and Ca decreased to more physiological levels. Bot use HCO.sub.3 /CO.sub.2. Both lack the proper NaCl ratio. (22) Earle WR. et al. J Nat'l Canc Inst 4, 165-212, 1943. Used with 5% CO.sub.2 and 20% O.sub.2. (23) Williams GM et al. Exp Cell Res 69, 106-112, 1971. (24) Hanks JH, Wallace RE. Proc Soc Exp Biol Med 71, 196 1949. For use outside CO.sub.2 incubators. (25) Ham RG. Proc Nat'l Acad Sci U.S. 53, 288, 1965. Analogous to Tyrode' (9) Table I. HCO.sub.3 deficient. (26) Delbecco R, Vogt M. J Exp Med 99, 167-182, 1954. Simply Krebs Ringer Phosphate with lowered Ca and Mg. The high Pi would lower cellular [.SIGMA. ATP]/[.SIGMA. ADP] [.SIGMA. Pi]. (26a) Delbecco R. Virology 8, 396, 1959. Lacks redox balance as does (22) and (25). The pH is high for general use.
Contemporarily, a large number of different aqueous electrolyte solutions or their salt concentrates are prepared sold in commerce, and used in in vitro fluids, principally as tissue culture fluid media.
Even a cursory examination of Table I will confirm the medical dicta that "plasma is an unmakable solution". The solutions listed in Table I illustrate this belief. The essential problem is that plasma contains, in addition to major inorganic electrolytes, trace quantities of various electrolytes plus various metabolites including plasma proteins. In practice, it has not been possible to construct synthetically a replication of plasma extracellular fluid or intracellular fluid because of their complexity. Blood, extracellular and intracellular fluid, and even plasma can be regarded as tissues.
In most prior art electrolyte solutions, the concentration of chloride anions (Cl.sup.-) is higher than in human plasma or serum. For example, the Krebs-Henseleit solution (see Table I) contains a concentration of Cl.sup.- which is about 20% higher than in fluids such as plasma. This anion gap, that is, the difference between the positive cations and the negative anions, is now known to be due largely to the anionic metabolites such fluids plus the contribution of acidic amino acid groups found on plasma proteins. Referring to Table I, it is seen that the total positive cations in, for example, human plasma is 142-154 meq/l while the total anions is only about 128-137 meq/l leaving a deficit of about 14-17 meq/l of anions. For convenience, the anion gap in such fluids can be expressed as the ratio of sodium cation milliequivalents per liter to chloride anion milliequivalents per liter.
From Table I, it is clear that the Kreb's Serum substitute (Kreb's, H. A. Biochem. Biophys. Acta 4, 249-269, 1950) comes closest to approximating the electrolyte composition of such fluids. In such solution, Krebs attempted to correct the excessive Cl.sup.- content in Krebs Henseleit solution (Hoppe. Z. Phusiol. Chem. 210, 33-66, 1932) using metabolic experiments with tissue slices. Because of the law of electrical neutrality, Na.sup.+ cannot be added to a solution without some anion (such as Cl.sup.-) being added also; the sum of cations and anions must be equal in any solution. In his 1950 attempt, Krebs chose pyruvate.sup.-, L-glutamate.sup.-, fumarate.sup.2- as anions to be added.
The alternative to Krebs-Henseleit is essentially Krebs-Ringers Phosphate or Delbecco's tissue culture media where Pi is present in amounts about 10 to 25 times normal plasma concentrations. Such media are used so as to eliminate HCO.sub.3 .sup.- /CO.sub.2. Both such solutions, used respectively in perfusion or cell culture not only have too high Pi which induces an abnormal intracellular [.SIGMA.ATP]/[.SIGMA.ADP]/[.SIGMA.Pi] ratio but also have too low a Na: Cl ratio inducing hyperchloremic acidosis.
The alternate use of lactate.sup.- or pyruvate alone induces severe abnormalities in cellular redox state and phosphorylation potential. The use of gluconate.sup.- induces abnormalities in the hexosemonophosphate pathway. Indeed, all previously used organic ions violate the "safe entry points" or the normal Na:Cl ratio as herein defined.
In addition to the use of lactate, gluconate, fumarate, glutamate, pyruvate, and citrate anions in current commercially available prior art electrolyte fluids, and wherein such anions are typically employed at levels above those found in the (plasma or serum) of healthy humans, many such prior art commercial fluids also employ high levels of nonionic metabolites, such as fructose and glycerol, which induce separate redox state and phosphorylation potential abnormalities of their own. Thus, fructose causes severe abnormalities in phosphorylation potential with rapid destruction of liver purine nucleotides and their release into blood sometimes leading to renal shutdown due to uric acid deposition in the kidneys (see Woods, H. F., Eggleston, L. V., and Krebes, H. A. Biochem. J. 119, 501-510, 1970). Fructose in plasma above 0.2 mM must be considered to violate the "safe entry point". Likeiwse, use of intravenous glycerol at levels above 5 mM/l as currently practiced leads, in tissue containing glycerol kinase, such as kidney and liver, to accumulation of 10 mM glycerol phosphate (over 100 times normal). (See Burch, H. B. et al. J. Biol. Chem. 257, 3676-3679, 1982).
Mammalian systems normally operate at temperatures between about 37.degree.-38.degree. C. whereas, by common thermodynamic convention, neutral pH is taken to be about 7 at 25.degree. C. It is clear that changes in pH, (the negative log.sub.10 of [H.sup.+ ] concentration) necessarily affect the fundamental energetic relationships occurring in living cells. Also, enzymes have sharply defined ranges of [H.sup.+ ] concentration in which they perform their catalytic functions in a normal manner. Deviation of mammalian plasma pH down to 6.9 or above 7.7 from its normal range of 7.35-7.45 is therefore fatal to most mammalian organisms. Massive changes in the cellular redox and phosphorylation states also disorder cellular homeostasis.
The pH of human plasma is normally maintained by the human body in the range from about 7.35 to 7.45 while the pH of human cellular cytoplasm is about 7.2 (see Veech et al. in J. Biol. Chem. 254, 6538-6547, 1979). If blood pH drops to 6.8 in man, then death ensues from cardiac arrest, and if blood pH increases to above pH 7.7, then death ensues from convulsions.
The major chemical system maintaining body pH within this narrow normal range is the [CO.sub.2 ]/[HCO.sub.3.sup.- ] buffer system. The [CO.sub.2 ] of blood is maintained minute to minute by a portion of the mammalian brain called the respiratory center which senses brain cell pH and adjusts the depth and speed of respiration in response to changes in pH by increasing or decreasing [CO.sub.2 ] according to the famous Henderson Hasselbalch equation (Henderson, L. J. Silliman Lectures, Yale U. Press, New Haven, 1928).
Even though pH is thus seen to be a critical factor in mammalian blood, many commercial electrolyte solutions used in vitro attempt to maintain pH with phosphate or even artificial buffers such as Tris and the like. The absence of CO.sub.2 /HCO.sub.3.sup.- necessarily induce profound changes in the [NADP.sup.+ ]/[NADPH] redox state but in all the metabolites of glycolysis (see Miller, A. L. et al. J. Neurochem. 25, 553-558, 1975).
The compositions and methods of the present invention overcome the above indicated prior art problems. These compositions and methods employ definite ratios of [bicarbonate.sup.- ]/[carbon dioxide], [1-lactate.sup.- ]/[pyruvate.sup.- ], and [d-betahydroxybutyrate.sup.- ]/[acetoacetate.sup.- ]. Each of these mixtures constitute a near equilibrium couple which is known to be a normal constituent of mammalian plasma. While each of these pairs of components has been previously employed at least on a laboratory basis in solutions used for animal (mammalian) experiments, these mixture pairs have never previously been used in an electrolyte solution to obtain a normal Na:Cl milliequivalent ratio or to solve the anion gap problem.
All previous electrolyte solutions, and plasma substitutes, induce severe and measurable pathogenic abnormalities and no prior art electrolyte solution or plasma substitute has both (a) employed at least one of the three mixture pairs of this invention and (b) achieved a normal Na:Cl milliequivalent ratio as taught herein. Thus, for example, the krebs-Henseleit solution contains the [HCO.sub.3.sup.- ]/[CO.sub.2 ] buffer system (but contains excessive chloride ions9. Schimassek (Schimassek, H. Bio. Chem. Z. 336, 460, 1963) added about normal blood levels of lactate and pyruvate to what is essentially Tyrode's solution (see tyrode, M. J. Arch. Int. Pharmacodyn 20. 205, 1910) containing a 2.5% albumin in an attempt to create a physiological solution for perfusion. It should be noted that Schimassek added 1.33 mM/L D-L-lactate, which is definitely abnormal (see normal blood lactate levels shown in Table I). Further, the Na.sup.+ of 151 mM/l ad Cl.sup.- of 147.5 mM/l in Schimassek's modified Tyrode's solution approximates the concentration of 155 mM/l Na and 155 mM/l Cl is so-called normal (0.9%) saline, the most widely used electrolyte infusion solution, and thus obtained a grossly abnormal Na:Cl milliequivalent ratio of 1.00. Normal plasma has a Na:Cl milliequivalent ratio of about 1.24-1.45 with a mean of about 1.38. Infusions of electrolyte solutions with a Na:Cl milliequivalent ratio of less than about 1.38 have long been known to cause hyperchloremic acidosis in the treated organism. (See Levinsky, N. G. in Harrison's Textbook of Medicine pp. 230-236, McGraw-Hill, N.Y., 1983). It is the attempt to avoid this problem that leads to the wide use of such solutions as Ringer's lactate or acetate dialysis fluids which overcome the Na:Cl ratio problem, but which in turn create gross abnormalities of other types. It is the attainment of a normal Na:Cl milliequivalent ratio in a manner which avoids the pathological consequences inherent in all currently known or practiced methods which is a major part of the invention herein disclosed.
The making of a Krebs-Henseleit electrolyte solution (or other prior art electrolyte solution) and the incorporation there into of a mixture of 1-lactate and pyruvate anions, or of a mixture of d-betahydroxybutyrate and acetoacetate anions did not, and could not, result in the making of an electrolyte solution wherein the anion gap problem was overcome (or wherein the milliequivalent ratio of sodium cations to chloride anions was normalized), in accordance with the teachings of the present invention, because each of such resulting solutions would still contain excessive chloride anions and so would inevitably cause hyperchloremia if and when used under in vitro use conditions.
In general summary, the prior art describes a series of electrolyte solutions typically of about 270-320 milliosmoles (or higher) comprised of: (a) 1 to 4 metallic cations of sodium, potassium, magnesium, and calcium in amounts greater than 0.5 mL/L, (b) 1 to 5 inorganic anions of chloride plus also H.sub.2 PO.sub.4.sup.2- HPO.sub.4.sup.1- (the later also called Pi herein), sulphate (SO.sub.4.sup.2-), (c) 0 to several organic carboxylic or bicarbonate anions, (d) 0 to about 12 nonionic materials in concentrations of greater than about 0.5 mL/L from the group comprising CO.sub.2 gas, glucose, urea, glutamine, and others, and (e) sometimes one or more high molecular weight substances, such as albumin, hemocel, and the like. None of these solutions, for the reasons herein above explained, either normalize the milliequivalent ratio of Na:Cl at all, or normalize this ratio without causing profound and adverse physiological consequences. In the present invention, there are provided processes and compositions of a complex fluid nature for in vitro usage which can substantially completely eliminate all of such prior art problems. While the components of these new solution compositions are known solution components, no one has heretofore formulated the solutions of the present invention which not only tend to achieve a normal plasma milliequivalent ratio of sodium cations to chloride anions, but also tend to achieve a normalization of plasma pH and a normalization of the cellular redox state and the cellular phosphorylation potential. Also, these new solutions permit one to avoid usage of the previously employed carboxylic anions, such as acetate, or lactate alone, which cause adverse effects.